An optimized method for 3D fluorescence co-localization applied to human kinetochore protein architecture
Two-color fluorescence co-localization in 3D (three-dimension) has the potential to achieve accurate measurements at the nanometer length scale. Here, we optimized a 3D fluorescence co-localization method that uses mean values for chromatic aberration correction to yield the mean separation with ~10...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2018-01-01
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| Series: | eLife |
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| Online Access: | https://elifesciences.org/articles/32418 |
| _version_ | 1818024058003914752 |
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| author | Aussie Suzuki Sarah K Long Edward D Salmon |
| author_facet | Aussie Suzuki Sarah K Long Edward D Salmon |
| author_sort | Aussie Suzuki |
| collection | DOAJ |
| description | Two-color fluorescence co-localization in 3D (three-dimension) has the potential to achieve accurate measurements at the nanometer length scale. Here, we optimized a 3D fluorescence co-localization method that uses mean values for chromatic aberration correction to yield the mean separation with ~10 nm accuracy between green and red fluorescently labeled protein epitopes within single human kinetochores. Accuracy depended critically on achieving small standard deviations in fluorescence centroid determination, chromatic aberration across the measurement field, and coverslip thickness. Computer simulations showed that large standard deviations in these parameters significantly increase 3D measurements from their true values. Our 3D results show that at metaphase, the protein linkage between CENP-A within the inner kinetochore and the microtubule-binding domain of the Ndc80 complex within the outer kinetochore is on average ~90 nm. The Ndc80 complex appears fully extended at metaphase and exhibits the same subunit structure in vivo as found in vitro by crystallography. |
| first_indexed | 2024-12-10T03:54:10Z |
| format | Article |
| id | doaj.art-dc0f3815e198472cacfc226dafe847a9 |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-12-10T03:54:10Z |
| publishDate | 2018-01-01 |
| publisher | eLife Sciences Publications Ltd |
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| series | eLife |
| spelling | doaj.art-dc0f3815e198472cacfc226dafe847a92022-12-22T02:03:10ZengeLife Sciences Publications LtdeLife2050-084X2018-01-01710.7554/eLife.32418An optimized method for 3D fluorescence co-localization applied to human kinetochore protein architectureAussie Suzuki0https://orcid.org/0000-0001-7390-5116Sarah K Long1Edward D Salmon2Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, United StatesDepartment of Biology, University of North Carolina at Chapel Hill, Chapel Hill, United StatesDepartment of Biology, University of North Carolina at Chapel Hill, Chapel Hill, United StatesTwo-color fluorescence co-localization in 3D (three-dimension) has the potential to achieve accurate measurements at the nanometer length scale. Here, we optimized a 3D fluorescence co-localization method that uses mean values for chromatic aberration correction to yield the mean separation with ~10 nm accuracy between green and red fluorescently labeled protein epitopes within single human kinetochores. Accuracy depended critically on achieving small standard deviations in fluorescence centroid determination, chromatic aberration across the measurement field, and coverslip thickness. Computer simulations showed that large standard deviations in these parameters significantly increase 3D measurements from their true values. Our 3D results show that at metaphase, the protein linkage between CENP-A within the inner kinetochore and the microtubule-binding domain of the Ndc80 complex within the outer kinetochore is on average ~90 nm. The Ndc80 complex appears fully extended at metaphase and exhibits the same subunit structure in vivo as found in vitro by crystallography.https://elifesciences.org/articles/32418Super-resolution light microscopycell divisionmitosiskinetochoreprotein architecture |
| spellingShingle | Aussie Suzuki Sarah K Long Edward D Salmon An optimized method for 3D fluorescence co-localization applied to human kinetochore protein architecture eLife Super-resolution light microscopy cell division mitosis kinetochore protein architecture |
| title | An optimized method for 3D fluorescence co-localization applied to human kinetochore protein architecture |
| title_full | An optimized method for 3D fluorescence co-localization applied to human kinetochore protein architecture |
| title_fullStr | An optimized method for 3D fluorescence co-localization applied to human kinetochore protein architecture |
| title_full_unstemmed | An optimized method for 3D fluorescence co-localization applied to human kinetochore protein architecture |
| title_short | An optimized method for 3D fluorescence co-localization applied to human kinetochore protein architecture |
| title_sort | optimized method for 3d fluorescence co localization applied to human kinetochore protein architecture |
| topic | Super-resolution light microscopy cell division mitosis kinetochore protein architecture |
| url | https://elifesciences.org/articles/32418 |
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